It has been estimated that 3% of the word population is infected with hepatitis C virus (HCV) and approximately 180 million individuals are chronic carriers of HCV. Recent studies have identified a number of neutralizing epitopes on the HCV E1 and E2 proteins and demonstrated with atomic structures how broadly neutralizing antibodies (bnAbs) interact with these epitopes. The 2.65-resolution structure of HCV E2 protein in complex with a bnAb (AR3C) revealed atomic details of this long-sought target, providing a structural basis for rational vaccine design. In this R21 application, we propose to combine the latest findings in HCV structural biology and cutting-edge technologies in protein design and next-generation sequencing (NGS) of antibody repertoires to facilitate HCV immunogen design for the induction of bnAbs in vaccination. The specific aims are: (1) to develop epitope-focused immunogens. The structures of an E1 and two E2 neutralizing epitopes in complex with bnAbs have been determined recently. We hypothesize that by grafting these linear epitopes onto protein scaffolds we can present these epitopes in their bnAb-bound conformations to induce cross-neutralizing antibodies. The epitope-scaffold antigens will be designed by a computational approach using an automated meta-server method to identify diverse protein scaffolds for optimal epitope grafting. Successfully designed and expressed antigens will be displayed in a multivalent manner (24 copies) on ferritin particle. (2) to engineer and optimize E2 glycoprotein as a subunit immunogen. We hypothesize that an optimized E2 protein with stabilized conformation can serve as an immunogen to elicit cross-neutralizing antibodies to multiple epitopes presented on the E2 surface. Based on the crystal structure of E2 core domain (E2c), we will engineer the variable loops to minimize immunodominant non-neutralizing antibody response, reengineer N- linked glycans on the surface, and introduce space-filling mutations in the front layer region to stabilize the exposed neutralizing face of E2c. Promising antigen candidates will be studied in immunization experiments and the antibody responses will be monitored by an array of immunological assays and by NGS-based antibody repertoire analysis.